DEVELOPING limited proteolysis and MASS SPECTROMEtry FOR THE CHARACTERIZATION of RIBOSOME TOPOGRAPHY

Moo-Jin Suh†, Soheil Pourshahian and Patrick A. Limbach*

Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, PO Box 210172, University of Cincinnati, Cincinnati, OH 45221

† Current address: Department of Pharmacology, Weill Medical College, Cornell University, New York, NY 10021

*To whom correspondence should be addressed.

SUPPLEMENTAL INFORMATION

Table S1.MALDI-TOFMS analysis of in-gel trypsin digestion of 1-D PAGE bands annotated in Figure S2. The identification of in-gel digestion is carried out using a 50 ppm mass tolerance with Protein Prospector.

Annotated
gel band / Matching
peptides / Sequence
Coverage / Accession
Number / Identity / Molecular Weight
(Da)
Intact proteins / A / 35 / 57% / P02349 / S1 / 61158
B / 12 / 49% / P02387 / L2 / 29729
C / 9
7 / 56%
36% / P02351
P02384 / S2
L1 / 26612
24598
D / 18 / 62% / P02352 / S3 / 25852
E / 22 / 80% / P02386 / L3 / 22244
F / 19 / 79% / P02354 / S4 / 23338
G / 18 / 70% / P02388 / L4 / 22087
H / 23 / 70% / P02390 / L6 / 18773
I / 24 / 87% / P02389 / L5 / 21070
J / 17
5 / 88%
37% / P02410
P02359 / L13
S7 / 16019
19888
Proteolytic fragments / O / 33 / 75% / L2
P / 28 / 70% / L2
Q / 18 / 62% / L3
R / 20 / 74% / S4
S / 19 / 60% / L4

Figure S1.MALDI mass spectral data after incubation of intact E. coli 70S ribosomes with Proteinase K under limited proteolysis conditions for the time periods denoted. The intact ribosomal proteins detected after each incubation period are summarized in Figure 3.

Figure S2.The 12.5% SDS PAGE results arising from the limited proteolysis of intact ribosomes using Proteinase K. M: protein marker; Lanes 1-9: intact ribosomes incubated with trypsin for 0 min, 30 min, 60 min, 125 min, 250 min, 500 min, 1000 min, 1500 min and 1500+60 min respectively. Identifications of the annotated bands A-J and O-S are presented in Table S1.

Figure S3.Representative MALDI mass spectral data after incubation of intact T. thermophilus 70S ribosomes with Proteinase K under limited proteolysis conditions for 1500 min. The intact ribosomal proteins detected after various incubation period are summarized in Figure S4.

Figure S4.Intact T. thermophilus ribosomal proteins from the (a) 30S subunit and (b) 50S subunitobserved with MALDI-MS after incubation with protease for denoted time periods. (white bars) Trypsin and (black bars) Proteinase K.

Figure S5.Tryptic fragments from T. thermophilus ribosomal protein L9 detected during the limited proteolysis studies described in the manuscript. (a) Placement of L9 in the T. thermophilus large subunit structure (pdb accession number 2J01). The first cleaved site (Lys 56) on L9 is indicated with an arrow. (b) The proteolytic domains tracked during 1500 min incubation. Only N-terminal domain (1-50) is detected after 1500 min. (c) Summary of tryptic fragments (sequence and mass) detected during the limited proteolysis.

Figure S6.Tryptic fragments from T. thermophilus ribosomal protein S18 detected during the limited proteolysis studies described in the manuscript. (a) Placement of S18 in the T. thermophilus small subunit structure (pdb accession number 2J00). One of the cleavage sites on S18 is indicated with an arrow. (b) The proteolytic domains tracked during 1500 min incubation. Only C-terminal domain (24-88) is detected after 1500 min. (c) Summary of tryptic fragments (sequence and mass) detected during the limited proteolysis.